This invention relates to newly identified auxotrophs, polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been putatively identified as being very important to the growth and/or reproduction of Aspergillus fumigatus. 
Generally, such proteins are of such importance to the growth and/or reproduction of Aspergillus fumigatus that modifications to the protein or to the polynucleotide encoding same, blocking the expression or activity of the protein, or deleting or disabling the polynucleotide encoding the protein will have a significant and clearly observable effect on either the growth or reproduction of the organism in vitro. In fact, absent a supplemented media having a particular substance that would have resulted from the synthesis pathway in which the protein functions, the Aspergillus fumigatus auxotrophs will die.
In accordance with one aspect of the present invention there are provided auxotrophic microbes of the Aspergillus fumigatus type, which are incapable of growth and reproduction in vitro in the absence of a media supplemented by at least one chemical compound that is not required for a non-auxotrophic microbe of the Aspergillus fumigatus type.
In accordance with another aspect of the present invention, there are provided novel proteins, as well as active fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the proteins of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such proteins.
In accordance with another aspect of the present invention there are provided strains of auxotrophic Aspergillus fumigatus microbe which have the ATCC Deposit Nos. AFH153 209347, AFLEU2 209348, and AFADE2 209349.
In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding mature polypeptides expressed by the DNA contained in ATCC Deposit Nos. AFH153 209347, AFLEU2 209348, and AFADE2 209349.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such proteins to produce antibodies specific for such proteins to permit analyzing a vector or host cell for the presence of the protein, which is heterologous to said vector or host cell. Thus, the protein is useful as a heterologous marker wherein the polynucleotide sequence encoding the protein is part of a construct inserted into a vector or host wherein such protein would be heterologous.
In accordance with a still further aspect of the invention another process utilizes the polynucleotides to assay for compounds which bind said polynucleotides and would thus block expression of any products from said polynucleotides.
In another aspect polynucleotides of the invention may be employed as a tool for studying Aspergillus fumigatus to ascertain various genes thereof, particularly other essential genes. One such process is useful for analyzing for the functionality of an unknown function cDNA from an Aspergillus fumigatus cDNA library comprising obtaining an auxotrophic strain of Aspergillus fumigatus, obtaining a polynucleotide construct comprising (i) a polynucleotide sequence capable of removing the auxotrophic property and (ii) at least one portion of the unknown function cDNA polynucleotide sequence. which is not the complete cDNA sequence from the cDNA library, and inserting said construct into said auxotrophic strain. Preferably, such a process involves an auxotroph that requires either histidine, adenylic acid, or leucine to grow and reproduce. A further preferred process, comprises assaying the auxotrophic strain for growth and reproduction in a media which lacks, histidine, adenylic acid, or leucine to confirm insertion of the construct. An ever further preferred process also comprises assaying the strain with the insertion for a lost property, which would have resulted from the unknown cDNA corresponding to the cDNA of the cDNA library.
Aspergillus fumigatus are microbes which are useful as host cells for the expression of heterologous polynucleotide sequences and for production of heterologous proteins. It would be helpful in such an environment to map more or all of the genes in This microbe in order to enhance its use as a host cell for expression of heterologous polynucleotide sequences and for production of heterologous proteins. Auxotrophs are useful in that they need a specific supplement in their media or they don""t grow or reproduce, and in fact may die. Thus, advantageously, a construct is made which comprises either the head or tail portion (preferably at least 250 base pairs in length) of the polynucleotide sequence that will cure (remove) the auxotrophic property ligated to the heterologous gene which is in turn ligated to the full polynucleotide sequence which will remove the auxotrophic property. Preferably, the construct may comprise a promoter sequence or a secretion coding sequence for the heterologous gene. Therefore, if a heterologous polynucleotide construct is inserted into the auxotroph which includes the gene encoding a protein or polypeptide (preferably according to this invention) which will eliminate the need for the supplement in the media, the auxotrophs can be conveniently screened for the successful insertion of the construct.
After attempts to insert the construct by homologous recombination (cross-over) in the auxotroph, the potential transformants are plated on supplemented media to culture colonies from a single isolated cell. Cells from a particular colony can then be plated on a media which lacks the supplement required by the starting auxotroph, species where insertion has been successful will grow on the media lacking that supplement, but species lacking the insert will fail to grow or reproduce and in fact may die. Accordingly, auxotrophs are useful tools to screen for successful insertion of heterologous genes which are part of a construct that removes the auxotrophic property of the auxotroph.
Such auxotrophs and the polynucleotides which encode for a protein which will remove a particular auxotrophic property of the auxotroph are also useful tools in the study of the genus or species of microbe from which the auxotroph is obtained. A vector containing a cDNA of unknown function from a cDNA library for the microbe may be utilized to form a construct having only a portion of the cDNA and including the known polynucleotide encoding the known protein which will eliminate the auxotrophic property of the auxotroph. The active gene corresponding to the cDNA from the library is disabled by successful insertion of the construct. For example, the culture is screened for successful insertion of the construct as discussed above, in a media which is fully supplemented except for the supplement required by the starting auxotroph. Species having the successful insertion, may then be studied to obtain the property of the disabled gene corresponding to the cDNA. For example, supplements may be individually omitted from the growth media until an effect is observed such as diminished growth or death, and the area of functionally of the gene corresponding to the cDNA is thus identified.
Accordingly, a process to determine the function of unknown genes within Aspergillus fumigatus utilizing the polynucleotides and/or proteins of the present invention is also an important and useful procedure made possible by the present invention.
There are other applications for the proteins and polynucleotides of the present invention in various industries which may utilize such microbe, such as in the fermentation industry. Since such proteins and/or polynucleotides have been found to be significantly essential to the growth and/or reproduction of Aspergillus fumigatus they may be useful to determine agonist which may enhance the growth and/or reproduction of such microbe in such fermentation processes. Moreover, the expression products of the polynucleotides according to the invention may be useful to enhance the growth of such microbe, e.g., multiple copies of the present gene in Aspergillus fumigatus may prove to enhance its growth or reproductive rates.
In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such proteins, or polynucleotides encoding such proteins, for purposes related to scientific research, for example, to generate probes for identifying similar sequences which might encode similar proteins from other organisms by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.